Over the past decade a great deal of research has been focused on the development of technology related to micro-total-analytical systems. This technology is based on the concept of a series of microfluidic channels also known as microchannels for the movement, separation, reaction, and/or detection of various chemicals or biological compounds such as amino acids, proteins, and DNA.
One disadvantage with prior microfluidic devices is that there is frequently a mismatch between the extremely small quantities of sample used for analysis and the often much larger quantities needed for loading the sample into the microfluidic device and transporting the sample to the point of analysis. For example, a typical analysis sample may be around one nanoliter or less of a liquid containing sample that is injected into a separation channel and then separated electrokinetically as it moves down the channel to a detection region. However, the channels used to transport the sample to the injection point are typically also filled with the sample, thus increasing the required amount of the sample by a factor of 100 or more. In addition, the sample is typically loaded onto the microfluidic device into a reservoir from a pipette so that in all, approximately 99.9% of the sample is discarded as waste.
Electric field gradient focusing is one way of addressing the problem of requiring a large sample for analysis due to the inefficiencies of conventional devices which result in wasted sample. Electric field gradient focusing can be used to concentrate samples at a given point within a microfluidic device before the analysis step. Further, the electric field gradient can be used to concentrate all of the sample at the beginning of the separation channel so that very little of the sample would be wasted.
Electric field gradient focusing is accomplished by the application of an electric field gradient within a microchannel. In response to the electric field gradient, there is a corresponding gradient in the electrophoretic velocity of any ion within the microchannel. The total velocity of the ion is the sum of its electrophoretic velocity and the bulk fluid velocity. If these two components of the velocity are in opposite directions, they can be balanced so that the molecule will have zero total velocity.
When there is a gradient in the electrophoretic velocity, the balance between bulk and electrokinetic velocities can occur at a single point within the microchannel and therefore can result in focusing of ions at that point. Typically, the electric field gradient used in focusing is generated by the external manipulation of the electric field in the middle of the microchannel through the use of conducting wires, salt bridges, porous membranes, or other structures that will pass electric current but will restrict the flow of bulk fluid and analytes that are to be focused.
Several recent developments with regard to focusing methods in microfluidics, and in particular, the use of electric field gradients, have been made. A description of related methods of focusing can be found in C. F. Ivory, W. S. Koegler, R. L. Greenlee, and V. Surdigio, Abstracts of Papers of the American Chemical Society 207, 177-BTEC (1994); C. F. Ivory, Separation Science and Technology 35, 1777 (2000); Z. Huang and C. F. Ivory, Analytical Chemistry 71, 1628 (1999); W. S. Koegler and C. F. Ivory, Journal of Chromatography a 726, 229 (1996); and P. H. Ofarrell, Science 227, 1586 (1985), all of which are hereby incorporated by reference.
To illustrate the basic principles disclosed in these publications, reference is made to FIG. 1(a) which depicts a length of buffer-filled microchannel of constant cross-sectional area with an electrode, denoted 4, in the middle, and two further electrodes at each end, denoted 3 and 5, so that the voltages V1, V3 at the ends and the voltage V2 at the middle of the channel can be controlled. A single species of negatively charged analyte is present in a buffer that is provided to the microchannel. The electrical connection, represented as electrode 4, can be accomplished with a simple metal wire as depicted in FIG. 1(a), or through a more complicated structure consisting of additional fluid channels and porous membrane structures or salt bridges.
The electric field in the section 1, i.e., the channel between electrodes 3 and 4 is E1=(V2−V1)/(l/2) and the electric field in section 2, i.e., between electrodes 4 and 5, is E2=(V3−V2)/(l/2), where V1, V2, and V3 are the voltages applied to the three electrodes 3, 4, and 5, and l is the length of the microchannel. If, E1 differs from E2 as shown in FIG. 1(b), the electrophoretic velocity of the analyte in the channel, uEP, will be different in section 1 than in section 2. If an overall bulk fluid velocity, uB<0, is applied, e.g., either electro-osmotic or pressure-driven, the bulk fluid velocity must be the same, due to continuity, in all parts of the microchannel. The total velocity of the analyte, uT=uB+uEP, will then be the sum of the electrophoretic and bulk velocities, which can differ in section 1 from section 2.
The use of the microchannel device of FIG. 1(a) for focusing of the ions is illustrated in FIG. 2 where uT,1>0>uT,2, so that the ions flow into the middle from both directions and are thus focused in the middle of the channel near electrode 4.
One major drawback to electric field gradient focusing is that the microchannel device tends to be difficult to construct and that it requires the control of voltage on an additional electrode, e.g. 4 of FIG. 1(a), that is used to apply the electric field gradient. In addition, if electrodes are used to generate electric field gradients, unwanted chemical products will be generated electrochemically at the buffer-electrode interface. If the electric field gradient is produced through the use of a salt bridge or membrane, the electrochemical products can be avoided, however only chemical species that cannot pass through the membrane or salt bridge can be focused.
Two additional methods for concentrating a sample include sample stacking and field amplified sample injection in which a sample is concentrated as the sample crosses a boundary between low and high conductivity buffers. These methods can achieve preconcentration factors of 100 to 1000-fold although these methods require multiple buffers. Sweeping is yet another concentration method which is capable of a very high degree of sample concentration (e.g., up to 5000-fold), but is useful only for small hydrophoic analytes with a high affinity for a mobile micellular phase.
An additional technique for concentrating an ionic sample includes isoelectric focusing. Isoelectric focusing is commonly used for the concentration and separation of proteins and involves the focusing of analytes at their respective isoelectric points (pls) along a pH gradient.
Two examples of recent isoelectric focusing techniques are provided by U.S. Pat. No. 3,664,939 to Luner et al. and U.S. Pat. No. 5,759,370 to Pawliszyn. Both references relate to isoelectric focusing with pH gradients that are created by the application of a temperature gradient. The isoelectric focusing uses a pH gradient to focus analytes and in particular proteins, at their isoelectric points. The isoelectric point is the pH at which the analyte has zero electrophoretic mobility, i.e., approximately zero charge. pH gradients for isoelectric focusing are typically generated using ampholyte mixtures or immobilized ampholytes in gels. The two above referenced patents are included here as examples of prior art uses of temperature gradients for focusing. It is actually very unusual for isoelectric focusing to be done with a pH gradient generated with using a temperature gradient.
One disadvantage with isoelectric focusing is that it is limited in application because it can only be used with analytes with an accessible pl. Additionally, the concentration to which a protein can be focused with isoelectric focusing is severely limited due to the low solubility of most proteins at their pls.